The long-term goal of this proposal is a greater understanding of how eukaryotic cells function. Using the yeast Saccharomyces cerevisiae as a model eukaryotic cell, I propose to focus on one particular biochemical pathway, that of proline degradation, to study (1) the genetic regulation of the genes which code for the two enzymes in the pathway and (2) the process by which these proteins are transported from the cytoplasm where they are synthesized to the mitochondrion where they function. Both in vivo and in vitro methods will be used to study the genetic regulation of this pathway. Yeast mutants containing inducer-independent as well as non-inducible enzyme activities will be sought by various screening methods. Site-specific insertion and deletion mutagenesis will be carried out on the recently cloned put2 DNA which codes for P5C dehydrogenase, the second enzyme in the pathway. In addition, gene fusions, which have been a powerful tool in the study of both gene regulation and protein transport, will be constructed between the put2 gene and the lacZ gene of E. coli, which codes for the enzyme Beta-galactosidase. These fusions will be used to isolate regulatory mutations as well as mutations affecting the subcellular localization of P5C dehydrogenase, i.e. the signal sequence, processing pathway. Studies on the movement of P5C dehydrogenase from the cytoplasm to the mitochondrion will be initiated by purification of the enzyme and the generation of specific antibodies to it. These will be used to determine if the protein is made as a larger molecular weight precursor which is processed in transit. Immunoprecipitation and SDS-polyacrylamide gel electrophoresis will be used to identify protein transport mutants. Because such health problems as cancer, the autoimmune diseases and the inherited metabolic diseases may involve aberrant regulation or the absence of, or improper processing of, crucial enzymes; it is necessary that we understand how the eukaryotic cell functions. By studying a system which has nuclear, cytoplasmic and mitochondrial components I hope to add to our knowledge of eukaryotic cell processes and shed light on the communication which exists between the various compartments of the cell.